The goal of this study was to determine whether the αv
integrin is a potential imaging biomarker for the presence of increased numbers of osteoclasts, which are implicated in bone diseases such as inflammatory osteolysis and osteolytic bone metastases. Osteoclasts are known to have high concentrations of the αv
integrin, and bone resorption can be blocked by blocking αv
). To show a proof of principle, we set out to determine whether the PET agent 64
Cu-CB-TE2A-c(RGDyK) is taken up specifically by osteoclasts and whether pharmacologically induced osteolysis is specifically imaged using this agent.
The group of Chen et al. has previously reported an RGD peptide monomer, a dimer, and a tetramer, each conjugated to the chelator DOTA for radiolabeling with 64
). However, on the basis of previous studies, 64
Cu-CB-TE2A-c(RGDyK) was expected to show improved liver clearance, compared with that of 64
). Because biodistribution showed that PTH treatment had no effect on liver uptake, post-PET data from control and PTH-treated mice were averaged in order to increase sample size. Post-PET liver uptake (1.52 ± 0.13 [n
= 5]) decreased by 22% and 27% relative to that in 1-h biodistribution control and PTH mice, respectively (P
= 0.001). In contrast, liver uptake did not decrease significantly between 1 and 4 h after injection for 64
= 0.34) (34
). These data further validate CB-TE2A as a bifunctional Cu(II) chelator superior to DOTA.
Cell uptake studies clearly demonstrated specific uptake of 64
Cu-CB-TE2A-c(RGDyK) by osteoclasts but not BMMs. Interestingly, the calculated affinity of 64
Cu-CB-TE2A-c(RGDyK) was significantly lower for isolated αv
than for integrin on the surface of osteoclasts in culture. αv
can transition between an active and a basal conformational state with a different ligand in the osteoclast (37
). Clustering of αβ heterodimers also modulates ligand binding (38
). We propose that the activation state of a purified integrin may be different from that of an integrin on an intact cell membrane in culture. Consistent with our ex vivo data, studies of related peptides have shown inhibition of tumor cell or osteoclast adhesion to vitronectin- or serum-coated surfaces, with IC50
values in the low micromolar range (39
We observed a 2.6-fold increase in maximal osteoclast uptake, compared with nonspecific BMM uptake. This relatively low level of specific uptake by osteoclasts in culture would not support 64Cu-CB-TE2A-c(RGDyK) as an agent able to detect osteoclasts in vivo. However, increased uptake at the site of induced osteolysis was clearly visible on small-animal PET. In addition, the region of highest uptake within the calvarium on PET/CT appeared to correlate with the sagittal suture, where the highest number of osteoclasts were noted on histology. To the best of our knowledge, no quantitative measurements of RGD-binding sites (Bmax) on osteoclasts in culture or in vivo have been previously reported. Therefore, higher numbers of in vivo binding sites or higher-affinity binding sites on activated osteoclasts cannot be ruled out.
Biodistribution studies demonstrated a significant increase in calvarium uptake in PTH-treated mice relative to controls. PTH did not appear to increase 64
Cu-CB-TE2A-c(RGDyK) uptake in tissues other than the calvarium. However, blocking with c(RGDyK) resulted in reduced uptake in all tissues examined. Similar multiorgan blocking has been reported for a related tetrameric RGD peptide in a tumor xenograft model (35
). It is well known that RGD peptides do show low-level affinity for other integrins (15
). Therefore, blocking studies do not appear well suited to demonstrating binding specificity for RGD peptides. Interestingly, the greatest degree of blocking was observed in the target tissues calvarium and femur—as would be predicted on the basis of the presence of osteoclasts—as well as in the spleen, the site of high levels of αv
-positive macrophages. Still, we did observe a significant correlation between bone surface area covered by osteoclasts and 64
Cu-CB-TE2A-c(RGDyK) uptake in the calvarium, thus suggesting osteoclast-specific uptake in bone. This relationship was likely reduced in our study because we examined whole calvarium uptake despite the fact that only a small portion of the calvarium was composed of osteoclasts.
The ability to noninvasively detect osteoclasts could have a significant impact on the clinical management of bone metastases by facilitating earlier detection of new lesions and potentially earlier follow-up of treatment response. In addition, imaging osteoclasts might allow clinicians to predict which patients would be responsive to treatment with bisphosphonates, a mainstay in bone metastasis therapy.
Beyond utility in managing neoplastic disease, osteoclast imaging could also find a use in several nonneoplastic bone diseases. For example, osteoporosis, which affects a large proportion of Western postmenopausal women, occurs when osteoclast-mediated bone resorption exceeds bone formation, and antiosteoporosis therapy typically targets osteoclasts (40
). Disease progression is monitored by measuring bone density over a period of years using dual-energy x-ray absorptiometry (40
). Osteoclast imaging might allow early follow-up after treatment initiation to determine whether the dose should be increased or an alternative therapy should be tried.
Periarticular osteolysis, a crippling complication of rheumatoid arthritis, is also caused by exuberant osteoclast recruitment (41
). Early imaging of the joints of rheumatoid patients may allow selection of those prone to the development of bone destruction. The relevance of this approach is underscored by the fact that although anticytokine therapy is effective in arresting periarticular osteolysis, the therapy is not without risk. Thus, identifying individuals with early, clinically undetectable joint destruction would be of value.
These studies strongly support specific uptake of 64Cu-CB-TE2A-c(RGDyK) by osteoclasts both ex vivo and in vivo. This osteoclast-mediated uptake was visualized on small-animal PET images of pharmacologically induced osteolysis, validating 64Cu-CB-TE2A-c(RGDyK) uptake in a lytic bone lesion independent of tumor cells. Although some tumor cells do express αvβ3, we propose that imaging tumor cells in conjunction with osteoclasts would improve sensitivity for early detection, though it might reduce utility in predicting the bone metastasis response to bisphosphonates. Additionally, αvβ3 ligands of higher affinity, such as CB-TE2A–conjugated dimers or tetramers of c(RGDyK), will undoubtedly improve specific uptake in osteoclasts, compared with that obtained with the c(RGDyK) monomer. Future studies will address these questions by imaging osteoclasts in animal models of bone metastasis.